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A Recombinant Bisphosphoglycerate Mutase Variant with Acid

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A Recombinant Bisphosphoglycerate Mutase Variant with Acid Proc. Nati. Acad. Sci. USA Vol. 91, pp. 3593-3597, April 1994 Biochemistry A recombinant bisphosphoglycerate mutase variant with acid phosphatase homology degrades 2,3-diphosphoglycerate MARIE-CLAUDE GAREL, NICOLE AROUS, MARIE-CLAUDE CALVIN, CONSTANTIN TIGELIU CRAESCU, JEAN ROSA, AND RAYMONDE ROSA Institut National de la Sante et de la Recherche M6dicale, U.91, H6pital Henri Mondor, 94010 Cr6teil, France Communicated by M. F. Perutz, January 3, 1994 (receivedfor review September 6, 1993) ABSTRACT To date no definite and undisputed treatment His in order to facilitate the orientation and binding of the has been found for sickle cell anemia, which is characterized by phosphate group and consequently activate the phosphoryl polymerization of a deoxygenated hemoglobin mutant (HbS) transfer to water (2). giving rise to deformed erythrocytes and vasoocclusive com- Little is known about the three-dimensional structure and plications. Since the erythrocyte glycerate 2,3-bisphosphate particularly localization of the active site residues in acid (2,3-DPG) has been shown to facilitate this polymerization, one phosphatases. Conversely, the structure of the yeast PGM therapeutic approach would be to decrease the intraerythro- has been determined by x-ray diffraction analysis by using cytic level of2,3-DPG by increasing the phosphatase activity of crystals soaked in 3-phosphoglycerate (3-PG). This latter the bisphosphoglycerate mutase (BPGM; 3-phospho-D- enzyme shares 5Ow sequence identity with human BPGM glycerate 1,2-phosphomutase, EC 5.4.2.4). For this purpose, and catalyzes the same three reactions (synthase, mutase, we have investigated the role of Gly-13, which is located in the and phosphatase), although at substantially different rates. active site sequence Arg'-His"'-Gly"1-Glu12-Gly13 in human These activities are catalyzed at the unique active site of BPGM. This sequence is similar to the Arg-His-Gly-Xaa-Arg* BPGM. To date and in spite of recent crystallization of sequence of the distantly related acid phosphatases, which human BPGM (9), no crystallographic data have been ob- catalyze as BPGM similar phosphoryl transfers but to a greater tained for this enzyme. Consequently, the amino acids in- extent. We hypothesized that the conserved Arg* residue in volved in the active site ofhuman BPGM have been deduced acid phosphatase sequences facilitates the phosphoryl transfer. by comparison with the structure of the yeast PGM (8). Consequently, in human BPGM, we replaced by site-directed Amino acid residues of the active site were highly conserved mutagenesis the corresponding amino acid residue Gly'3 with between BPGM and PGM. The different catalytic rates an Arg or a Lys. In another experiment, we replaced Gly'3 with observed for these two homologous enzymes could be ex- Ser, the amino acid present at the corresponding position ofthe plained by the nonconserved residues in their active site. homologous yeast phosphoglycerate mutase (n-phosphoglycer- Among them, Gly13 in BPGM and the homologous Ser11 in the ate 2,3-phosphomutase, EC 5.4.2.1). Mutation of Gly'3 to Ser yeast PGM have been postulated to play an important role (7, did not modify the synthase activity, whereas the mutase and 8). the phosphatase were 2-fold increased or decreased, respec- Synthase and phosphatase activities of BPGM catalyze, tively. However, replacing Gly'3 with Arg enhanced phospha- respectively, the synthesis and degradation of glycerate tase activity 28.6-fold, whereas synthase and mutase activities 2,3-bisphosphate (2,3-DPG), the main allosteric effector of were 10-fold decreased. The presence of a Lys in position 13 hemoglobin. In spite of the presence of acid phosphatases in gave rise to a smaller increase in phosphatase activity (6.5-fold) erythrocytes, the degradation of2,3-DPG is very low because but an identical decrease in synthase and mutase activities. these acid phosphatases cannot degrade 2,3-DPG and the Taken together these results support the hypothesis that a phosphatase activity of BPGM is very slow (1000-fold lower positively charged amino acid residue in position 13, especially than the synthase activity and lower than that of acid phos- Arg, greatly activates the phosphoryl transfer to water. These phatases). Consequently, 2,3-DPG is present in high concen- results also provide elements for locating the conserved Arg* trations in erythrocytes. residue in the active site of acid phosphatases and facilitating Our purpose being to decrease 2,3-DPG level in erythro- the phosphoryl transfer. The implications for genetic therapy cytes by increasing the BPGM phosphatase activity, we have of sickle cell disease are discussed. compared amino acid sequences ofseveral acid phosphatases possessing a His residue in their active site and especially the Some enzymes displaying phosphatase activities such as sequences of a fragment common to these enzymes and human prostatic acid phosphatase (1, 2), lysosomal (3) and human BPGM (Fig. 1) (4). This fragment, corresponding to yeast (4) acid phosphatases, erythrocyte bisphosphoglycer- the amino acid sequence Arg9-His10-Gly11-Glu12-Gly13 in hu- ate mutase (BPGM; 3-phospho-D-glycerate 1,2-phosphomu- man BPGM, is homologous to the amino acid sequence tase, EC 5.4.2.4) (5-7), glycolytic phosphoglycerate mutase Arg7-His8-Gly9-Gln10-Ser'1 in yeast PGM, which is localized (PGM; D-phosphoglycerate 2,3-phosphomutase, EC 5.4.2.1) in the active site and contains the phosphorylatable His (7, 8, (7, 8), and hepatic 6-phosphofructo-2-kinase/fructose-2,6- 10). From this analysis, we have postulated that the con- bisphosphatase (fructose-2,6-bisphosphate 2-phosphatase; served Arg residue, denoted Arg* in the acid phosphatase D-fructose-2,6-bisphosphate 2-phosphohydrolase, EC consensus sequence and which corresponds to the Gly13 in 3.1.3.46) (1) possess a His residue in their active site, which BPGM, could be a good candidate as a cationic group for is transiently phosphorylated during the course of the phos- enhancing the phosphoryl transfer. We have therefore sub- phoryl transfer. It was suggested that most such enzymes stituted Arg for Gly13 in human BPGM by site-directed involved in the binding of phosphate esters should have at mutagenesis. We have also substituted Lys for Gly13 to least one cationic group suitably disposed near the active site evaluate the role of another positively charged amino acid at The publication costs of this article were defrayed in part by page charge Abbreviations: 2,3-DPG, glycerate 2,3-bisphosphate; 3-PG, glycer- payment. This article must therefore be hereby marked "advertisement" ate 3-phosphate; BPGM, bisphosphoglycerate mutase; PGM, phos- in accordance with 18 U.S.C. §1734 solely to indicate this fact. phoglycerate mutase. 3593 Downloaded by guest on October 2, 2021 3594 Biochemistry: Garel et al. Proc. NatL. Acad. Sci. USA 91 (1994) Hu-BPGM Arg9 His10 GIyll G1u12 G1y13 tion was performed on an HPLC column of Fractogel TSK AF blue (Merck) at room temperature as reported (17). HU-PGM-M Arg His Gly G1u Thr Enzyme and 2,3-DPG Assays. Synthase and mutase activ- Ye-PGM Arg7 His8 GIy9 Gin10 Ser11 ities were assayed according to methods already reported (18). When synthase activity was assayed directly on the E. F-2,6-P2ase Arg His Gly Glu Ser colicrude extracts, this assay being directly related to NADH production by glyceraldehyde phosphate dehydrogenase, the reaction could be partially masked by the intrinsic NADH Hu-P-ACP Arg His Gly Asp Arg* oxidase activity ofE. coli. It was, therefore, necessary to add 2 mg ofantimycin A and 1 mmol ofKCN to 1 ml ofthe assay Hu-L-ACP Arg His GIl ASP Arg* system as specific inhibitors of E. coli NADH oxidase. The Ye-ACP1 Arg His Gly Ser Arg* phosphatase activity was measured by coupling the reaction with phosphoglycerate kinase, glyceraldehyde phosphate Ye-ACP3 Arg His Gly Glu Arg* dehydrogenase, triosephosphate isomerase, and glycerol Ye-ACP5 Arg His Gly GIu Arg* phosphate dehydrogenase. In this sequence of reactions, the degradation of 1 mol of 3-PG is coupled with the oxidation of FIG. 1. Conserved peptide sequence in human BPGM (Hu- 2 mol of NADH. Each sample was checked against a control BPGM), human muscle PGM (Hu-PGM-M), yeast PGM (Ye-PGM), deprived of 2,3-DPG. In the case of 2-phosphoglycolate hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (F- stimulation of the phosphatase reaction, 1 mM 2-phospho- 2,6-P2ase), human prostatic acid phosphatase (Hu-P-ACP), human glycolate was added to the standard assay system and be- lysosomal acid phosphatase (Hu-L-ACP), yeast acid phosphatase P1 cause of its potent effect (19) incubation was performed for (Ye-ACP1), yeast acid phosphatase P3 (Ye-ACP3), and yeast acid 15 min instead of 60 min and 0.4 mg of the enzyme was used phosphatase PS (Ye-ACP5). instead of 4 mg. The amount of 2,3-DPG was measured this position in the active site. Another substitution was enzymatically on the deproteinized extracts by techniques performed by replacing Gly" with Ser to analyze the modi- previously reported (18), and its amount was related to the fications ofthe catalytic properties of BPGM when this Gly13 ratio ofprotein in the lysate measured according to Lowry et is replaced by the amino acid residue present in the homol- al. (20). ogous yeast PGM. Electrophoresis. PAGE was performed in the presence of SDS according to Laemmli (21). The gels were stained with Coomassie blue R250 and the amount of expressed wild-type MATERIALS AND METHODS or mutant BPGM was determined by densitometric scanning Materials. Except when specified otherwise the reagents of the gels. used for the buffers were obtained from Merck. All substrates Thermostability Studies. The purified variants were incu- and commercial enzymes were purchased from Boehringer bated at 550C for 30 min in 10 mM Tris HCl buffer (pH 7.5) Mannheim except for NADH, which was a product of Sigma containing 1 mM EDTA, 1 mM 2-mercaptoethanol, and 1 mg as were Trizma base (Tris), bovine serum albumin, and of bovine serum albumin per ml.
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